Leptospira interrogans thermolysin refolded at high pressure and alkaline pH displays proteolytic activity against complement C3

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dc.contributorLaboratório de Bacteriologiapt_BR
dc.contributor.authorChura-Chambi, Rosa Mariapt_BR
dc.contributor.authorFraga, Tatiana Rodriguespt_BR
dc.contributor.authorSilva, Ludmila Bezerra dapt_BR
dc.contributor.authorYamamoto, Bruno Bernardipt_BR
dc.contributor.authorIsaac, Lourdespt_BR
dc.contributor.authorBarbosa, Angela Silvapt_BR
dc.contributor.authorMorganti, Ligiapt_BR
dc.date.accessioned2020-07-09T21:20:57Z-
dc.date.available2020-07-09T21:20:57Z-
dc.date.issued2018-
dc.identifier.citationChura-Chambi RM, Fraga TR, Silva LB, Yamamoto BB, Isaac L, Barbosa AS, et al. Leptospira interrogans thermolysin refolded at high pressure and alkaline pH displays proteolytic activity against complement C3. Biotechnol Rep. 2018 Sep;19:e00266. doi:10.1016/j.btre.2018.e00266.pt_BR
dc.identifier.issn2215-017X-
dc.identifier.urihttps://repositorio.butantan.gov.br/handle/butantan/2533-
dc.description.abstractEnzymes from the thermolysin family are crucial factors in the pathogenesis of several diseases caused by bacteria and are potential targets for therapeutic interventions. Thermolysin encoded by the gene LIC13322 of the causative agent of leptospirosis, Leptospira interrogans, was shown to cleave proteins from the Complement System. However, the production of this recombinant protein using traditional refolding processes with high levels of denaturing reagents for thermolysin inclusion bodies (TL-IBs) solubilization results in poor recovery and low proteolytic activity probably due to improper refolding of the protein. Based on the assumption that leptospiral proteases play a crucial role during infection, the aim of this work was to obtain a functional recombinant thermolysin for future studies on the role of these metalloproteases on leptospiral infection. The association of high hydrostatic pressure (HHP) and alkaline pH was utilized for thermolysin refolding. Incubation of a suspension of TL-IBs at HHP and a pH of 11.0 is non-denaturing but effective for thermolysin solubilization. Soluble protein does not reaggregate by dialysis to pH 8.0. A volumetric yield of 46 mg thermolysin/L of bacterial culture and a yield of near 100% in relation to the total thermolysin present in TL-IBs were obtained. SEC-purified thermolysin suffers fragmentation, likely due to autoproteolysis and presents proteolytic activity against complement C3 a-chain, possibly by a generation of a C3b-like molecule. The proteolytic activity of thermolysin against C3 was time and dose-dependent. The experience gained in this study shall help to establish efficient HHP-based processes for refolding of bioactive proteins from IBs.pt_BR
dc.description.sponsorshipFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)pt_BR
dc.format.extente00266pt_BR
dc.languageengpt_BR
dc.publisherElsevierpt_BR
dc.relation.ispartofBiotechnology Reportspt_BR
dc.rightsOpen Accesspt_BR
dc.titleLeptospira interrogans thermolysin refolded at high pressure and alkaline pH displays proteolytic activity against complement C3pt_BR
dc.typeArticlept_BR
dc.identifier.doi10.1016/j.btre.2018.e00266pt_BR
dc.identifier.urlhttp://dx.doi.org/10.1016/j.btre.2018.e00266pt_BR
dc.contributor.externalInstituto de Pesquisas Energéticas e Nucleares (IPEN)¦¦Brasilpt_BR
dc.contributor.externalUniversidade de São Paulo (USP)¦¦Brasilpt_BR
dc.publisher.cityAmsterdampt_BR
dc.identifier.citationvolume19pt_BR
dc.subject.keywordThermolysinpt_BR
dc.subject.keywordMetalloproteasept_BR
dc.subject.keywordRefoldingpt_BR
dc.subject.keywordHigh hydrostatic pressurept_BR
dc.subject.keywordAlkaline pHpt_BR
dc.relation.ispartofabbreviatedBiotechnol Reppt_BR
dc.identifier.citationabntv. 19, e00266, set. 2018pt_BR
dc.identifier.citationvancouver2018 Sep;19:e00266pt_BR
dc.publisher.countryNetherlandspt_BR
dc.contributor.butantanSilva, Ludmila Bezerra da|:Aluno|:Laboratório de Bacteriologia|:pt_BR
dc.contributor.butantanYamamoto, Bruno Bernardi|:Aluno|:Laboratório de Bacteriologia|:pt_BR
dc.contributor.butantanBarbosa, Angela Silva|:|:Laboratório de Bacteriologia:Laboratório de Bacteriologia|:pt_BR
dc.sponsorship.butantanFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)¦¦2016/17534-6pt_BR
dc.sponsorship.butantanFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)¦¦2014/00926-3pt_BR
dc.sponsorship.butantanFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)¦¦2015/02574-0pt_BR
dc.sponsorship.butantanFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)¦¦2013/17419-4pt_BR
dc.identifier.bvsccBR78.1pt_BR
dc.identifier.bvsdbIBProdpt_BR
item.openairetypeArticle-
item.fulltextCom Texto completo-
item.grantfulltextembargo_29990101-
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